MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

酶学、动力学和合成、物理和分析化学 用于逆转录酶的机制研究和 人类免疫缺陷病毒1型(HIV-1)的蛋白酶，与 最终目标是为这些酶开发特定的抑制剂。i) 在之前的报告中，我们描述了蛋白质分解步骤的动力学。 从含有HIV-1蛋白酶的构建物中自动处理 蛋白水解酶序列由19个氨基酸组成的Pol序列位于C-端 末端和反式框肽的12个氨基酸序列位于 N-末端与E. Coli.我们现在已经通过荧光光谱学研究了 启动自动加工的融合蛋白的复性。二 过程被观察到：一个或多个初始步骤的速度太慢 使用传统混合技术测量速度较快，步骤较慢 这归因于多肽键的顺反异构化。 涉及到脯氨酸的亚氨基氮。后一步是大约15步。 比氨基末端的初始蛋白水解性裂解快几倍 蛋白水解酶。这一蛋白质分解步骤发生在 单独与二聚体融合蛋白分子内结合，以及 在成熟的HIV-1蛋白酶存在的分子间。尽管 二聚体融合蛋白催化活性低，具有相当高的催化活性 需要浓度为1.4fM的成熟蛋白酶才能给出一个速率 这与分子内切割是竞争的，因为 分子内性的熵优势。13.2 kDa的蛋白质 通过在氨基末端裂解而形成的中间体 蛋白水解酶序列经纯化后，同源性达90%。初步 结果表明，二聚体形式的离解常数 这种蛋白质比任何一种全长融合的蛋白质都要大得多 蛋白质或成熟的蛋白酶。Ii)非过程性递增的动力学 Klenow法将4-硫代胸腺嘧啶核苷转化为DNA模板-引物 测定了大肠杆菌DNA聚合酶I的片段 在335 nm处分光光度测定。离解速率常数 从酶中产生的寡核苷酸产物大约是它的6倍 比其含有胸腺嘧啶核苷的相应速率常数 模拟的。

Enzymology, kinetics and synthetic, physical and analytical chemistry are used in mechanistic investigations of the reverse transcriptase and protease of human immunodeficiency virus type 1 (HIV-1), with the ultimate goal of developing specific inhibitors for these enzymes. i) In a previous report, we described the kinetics of proteolytic steps in the autoprocessing of the HIV-1 protease from a construct containing the protease sequence flanked by a 19-amino acid Pol sequence at the C- terminus and a 12-amino acid sequence from the trans frame peptide at the N-terminus, which is fused to the maltose binding protein of E. coli. We have now examined, by fluorescence spectroscopy, the renaturation of the fusion protein that initiates autoprocessing. Two processes were observed: an initial step (or steps) whose rate was too fast to measure using conventional mixing techniques, and a slower step that was attributed to cis/trans isomerization of peptide bonds involving the imino nitrogen of proline. The latter step is ca. 15 times faster than the initial proteolytic cleavage at the amino terminus of the protease sequence. This proteolytic step occurs both intramolecularly with the dimeric fusion protein alone, and intermolecularly in the presence of mature HIV-1 protease. Despite the low catalytic activity of the dimeric fusion protein, a fairly high concentration (1.4 fM) of mature protease is required to give a rate that is competitive with intramolecular cleavage, as a result of the entropic advantage of intramolecularity. The 13.2-kDa protein intermediate that is formed by cleavage at the amino terminus of the protease sequence has been purified to >90% homogeneity. Preliminary results suggest that the dissociation constant for the dimeric form of this protein is much larger than that for either the full length fusion protein or the mature protease. ii) Kinetics of nonprocessive incor- poration of 4-thiothymidine into a DNA template-primer by the Klenow fragment of E. coli DNA polymerase I were measured spectrophotometrically at 335 nm. The rate constant for dissociation of the oligonucleotide product from the enzyme is ca. 6 times larger than the corresponding rate constant for its thymidine-containing analog.